Fuel-driven macromolecular coacervation in complex coacervate core micelles

Fuel-driven macromolecular coacervation is an entry into the transient formation of highly charged, responsive material phases. In this work, we used a chemical reaction network (CRN) to drive the coacervation of macromolecular species readily produced using radical polymerisation methods. The CRN enables transient quaternization of tertiary amine substrates, driven by the conversion of electron deficient allyl acetates and thiol or amine nucleophiles. By incorporating tertiary amine functionality into block copolymers, we demonstrate chemical triggered complex coacervate core micelle (C3M) assembly and disassembly. In contrast to most dynamic coacervate systems, this CRN operates at constant physiological pH without the need for complex biomolecules. By varying the allyl acetate fuel, deactivating nucleophile and reagent ratios, we achieved both sequential signal-induced C3M (dis)assembly, as well as transient non-equilibrium (dis)assembly. We expect that timed and signal-responsive control over coacervate phase formation at physiological pH will find application in nucleic acid delivery, nano reactors and protocell research.

DVP is a known compound and was synthesized following reported procedures 1, 2 (Scheme S).
Analysis by 1 H NMR spectroscopy demonstrated a noticeable shift in the aromatic pyridine signals, while a shift and broadening was observed for the DVP signals (see Figure S12). By quantitating these shifts, a 65% conversion to the cationic pyridine adduct in P1 (VPD + ) was determined after 5 days reaction (Figures S8 and S10). The TEM images proposed to contain micelles were analysed using Image J software by manually drawing spheres around 30 species per image to record an average diameter. Values reported in Note that spectra is cut into 3 rescaled segments for clarity. After 120 hours sample was mixed with various polyamines in the intial morphology study (Figure ). Note that spectra is cut into 3 rescaled segments for clarity. *Additional signals observed around 6.0 -6.5 ppm at 5 h are believed to be related to phosphate nucleophilic substitution onto VPM + (ME-PB, see Figure S18).  with ME as the allyl acetate, the same procedure was followed with DVP replaced with ME (2.5 mg, 16 µmol). For the DVP experiment conducted at 37.5°C (all other experiments at 25°C), heating was achieved in the DLS cell or by submerging the cuvette in a 38°C water bath. DLS data from the initial 25°C (room temperature) experiment with DVP is presented in Figure S15.
Additional DLS and TEM data for the dilute (4 mM) experiments is presented in Figures S16 -S17 (DVP) and Figures S22 -S24 (ME), including a repeat of the ME experiment demonstrating great reproducibility.      were identified during the reaction (ME-PB and ME-OH), believed to be the phosphate substitution and hydrolysis product of VPM + (see Figure S20 for further discussion and evidence).
Note that the spectra are cut into 3 rescaled segments for clarity. Polyanions excluded from this experiment to avoid supression of signals in micelles. The signal marked with (*) is believed to be due to methanol, which could arise from ester hydrolysis of many species in this reaction network. Figure S19. Additional 1 H NMR (D2O, pre-sat) spectra from ME and SH signal induced P1 (de)ionization experiment (See Figure 4 and Figure S18). After addition of a further SH (up to ~ 2.5 equivalents), complete reformation of the starting polyamine is demonstrated. The additional SH is required due to double Michael adduct (ME-2SH) formation, which after addition of excess SH is the main waste product (insignificant ME-SH remaining from 1 H NMR). Note that spectra are cut into 2 rescaled segments for clarity. Figure S20. 1 H NMR (D2O, + 100 mM PB buffer pH 7.4 where described) study of additional allylic species formed during ME fuelled P1 ionization. Additional species are proposed to be the phosphate substitution product (ME-PB) and the hydrolysis product (methyl 2-(hydroxymethyl)acrylate), ME-OH). a) ME-OH is commercially available (Fluro Chem) and a reference spectrum is presented. b) ME was combined with a polymer similar to P1 (p4VP104-b-DMA261) in the absence of PB buffer, spectra shown is after 120 h. c) ME was combined with p4VP104-b-DMA261 in PB buffer (30% D2O), spectra shown is after 120 h. d) ME was dissolved into PB buffer (30% D2O) alone, spectra shown is after 160 h. In all cases some hydrolysis was observed (3 -7% of ME after > 100 h). However only in cases where PB buffer was included is the doublet at 4.48 ppm observed (J= 6.5 Hz), along with allylic peaks at 6.33 and 6.05 ppm        NMR data presented in a) for conversion to VPM + is only an estimate obtained by assuming the difference between ME consumed and ME-SH + ME-SH2 produced equates to VPM + production since the signals for the species are supressed in the micelle core. Value is expressed as theoretical % of VP units on P1 converted to VPM + .

Figure S29
TEM images demonstrating extent of micelle re-assembly in transient C3M disassembly experiments (starting with 3 eq. of an allyl acetate) after 2.0 eq. of SH have been added a) with DVP as the allyl acetate micelle re-assembly appeared to be incomplete, even after at t = 1000 h. Spherical objects were measured as 13.8 ± 4.9 nm. b) with ME as the allyl acetate micelle assembly appeared to be successful at t = 65 h. Spherical objects were measured as 17.5 ± 2.7 nm.  Peaks labelled in red and blue were integrated to quantitate the extent of conversion to cationic pyridine adduct (VPD + ) in P1 and waste (DVP-Thr), respectively. Addition of PSS in experiment lead to supression of peaks related to VPD + due to inclusion in micelle core. Note that DVP-Thr contains a secondary amine known to also react with VPD + and although not identified here is likely also have formed. 5 Spectra is cut into 2 rescaled segments for clarity. [DVP] = 8 mM (addition at 10 h)). Note that spectra is cut into 2 rescaled segments for clarity.  In similar experiments in our laboratory we have found that addition of a biocide such as NaN3